Requirement of Phosphatidylinositol 3-Kinase Activity for Translocation of Exogenous aFGF to the Cytosol and Nucleus*
- From the Department of Biochemistry at The Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway
Abstract
Acidic fibroblast growth factor (aFGF) is a potent mitogen for many cells. Exogenous aFGF is able to enter the cytosol and nucleus of sensitive cells. There are indications that both activation of the receptor tyrosine kinase and translocation of aFGF to the nucleus are of importance for mitogenesis. However, the mechanism of transport of aFGF from the cell surface to the nucleus is poorly understood. In this work we demonstrate that inhibition of phosphatidylinositol (PI) 3-kinase by chemical inhibitors and by expression of a dominant negative mutant of PI 3-kinase blocks translocation of aFGF to the cytosol and nucleus. Translocation to the cytosol and nucleus was monitored by cell fractionation, by farnesylation of aFGF modified to contain a farnesylation signal, and by phosphorylation by protein kinase C of aFGF added externally to cells. If aFGF is fused to diphtheria toxin A-fragment, it can be artificially translocated from the cell surface to the cytoplasm by the diphtheria toxin pathway. Upon further incubation, the fusion protein enters the nucleus due to a nuclear localization sequence in aFGF. We demonstrate here that upon inhibition of PI 3-kinase the fusion protein remains in the cytosol. We also provide evidence that the phosphorylation status of the fusion protein does not regulate its nucleocytoplasmic distribution.
Footnotes
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↵* This work was supported by the Norwegian Cancer Society, Novo Nordisk Foundation, the Norwegian Research Council for Science and Humanities, Blix Legat, Rachel and Otto Kr. Bruun's Legat, and by The Jahre Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Postdoctoral fellow of The Norwegian Cancer Society.
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↵§ To whom correspondence should be addressed. Tel.: 47 22935640; Fax: 47 22508692; E-mail: olsnes@radium.uio.no.
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↵2 Klingenberg, O., Wiedtocha, A., Rapak, A., Khnykin, D., Citores, L., and Olsnes, S. (2000) J. Cell Sci.,in press.
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↵3 A. Wiedlocha, unpublished results.
- Abbreviations:
- aFGF
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acidic fibroblast growth factor
- FGFR
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FGF receptor
- DT
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diphtheria toxin
- DT-A
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DT A-fragment
- DT-B
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DT B-fragment
- NLS
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nuclear localization sequence
- PI
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phosphatidylinositol
- SH2-/SH3-domain
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src homology 2/3 domain
- DMEM
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Dulbecco's modified essential medium
- PAGE
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polyacrylamide gel electrophoresis
- PBS
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phosphate-buffered saline
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- Received January 21, 2000.
- The American Society for Biochemistry and Molecular Biology, Inc.
